Influence of iron deprivation and sub-inhibitory concentrations of antifungal antibiotics on surface antigens of candida albicans yeast cells

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

Studies of the stability and metabolism of tumour inhibitory tetrazinones

Temozolomide is an imidazotetrazinone with antineoplastic properties. It is structurally related to dacarbazine. Temozolomide was not metabolized in vitro by liver fractions. Chemical decomposition appears to play an important r^ole in its in vitro and in vivo disposition. In contrast, 3-methylbenzotriazinone, a structural analogue, was metabolized by hepatic microsomes to afford benzotriazinone and a hydrophilic metabolite. The cytotoxicity of temozolomide, dacarbazine, 5-[3-(hydroxy-methyl-3-methyl-triazen-1-yl]imidazole-5-carboxamide (HMMTIC) and 3-monomethyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were investigated in TLX5 murine lymphoma cells. Unlike dacarbazine, which was not toxic, MTIC, HMMTIC and temozolomide were cytotoxic in the absence of microsomes. Decarbazine was only cytotoxic in the presence of microsomes. The formation of MTIC from dacarbazine, HMMTIC and temozolomide was determined by reversed phase high performance liquid chromatography in mixtures incubated under conditions identical to those described before. MTIC was generated chemically from temozolomide and HMMTIC metabolically from dacarbazine. Using [14C]temozolomide, it was found that, in mice, the major route of excretion of the drug is via the kidneys. An acidic metabolite (metabolite I) was found in the urine of mice which had received temozolomide but its identity has not been established. 1H NMR, UV and chemical analyses revealed that Metabolite I possesses an intact NNN linkage and the site of metabolism is at the N3 methyl group. A further acidic metabolite (metabolite II) was found in the urine of patients. Metabolite II was unambiguously identified as the 8-carboxylic acid derivative of temozolomide. In vitro cytotoxicity assay showed that ony metabolite II is cytotoxic but not metabolite I. Pharmacokinetic studies of temozolomide and MTIC in vivo were performed on mice bearing TLX5 tumour. Temozolomide was eliminated from the plasma monophasically with a t1/2 of 0.7hr. MTIC was identified as a product of decomposition. MTIC was eliminated rapidly with a t1/2 of 2min. Though temozolomide shares many biochemical and biological similarities with clinically used dacarbazine, the results obtained in this study show that it differs markedly in its pharmacokinetic properties from dacarbazine, as temozolomide produced relatively sustained plasma levels which were reflected by drug concentrations in the tumour.

The functional effects of ceramide on the monocyte CD36 scavenger receptor and NADPH oxidase

Atherosclerosis is the principal cause of death in the United States, Europe and much of Asia. During the last decade, inflammation has been suggested to play a key role in the development of atherosclerosis. Reactive oxygen species (ROS) released during inflammation additionally oxidize LDL, which is subsequently taken up in an unregulated way through scavenger receptors on macrophages to form foam cells, the hallmark of atherosclerotic lesions. Previous work has shown that the lipid ceramide, which is found in aggregated LDL and in atherosclerotic plaques, decreases intracellular peroxide most likely through reducing NADPH oxidase activity. Ceramide is an important component of membrane microdomains called lipid rafts which are important for membrane protein function. Endogenous ceramide enhances lipid raft f'ormation and alters theirs composition. NADPH oxidase membrane subunits cytochrome b558 (which includes gp91) strongly associates with lipid rafts Therefore present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrurting the membrane component of NADPH oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flottillin and the raft environment. NADPH oxidase membrane component gp9J phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signaling pathways associated with atherogenesis from the activation of sphingomyelinase (SMase). It has been reported that SMase enhances LDL receptor mediated LDL endocytosis. However, no study has been done to investigate the effect of ceramide on scavenger receptors such as CD36 and oxidized LDL (OxLDL) uptake. CD36 is the major recertor far OxLDL. Reduced CD36 expression results in less foam cell formation and less atherosclerotic lesion without disrupting the clearance of OxLDL from plasma. This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLOL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLOL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of C036 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LOL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.

Polarized secretion of leukemia inhibitory factor

Background: The direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor LIF) belongs to the interleukin-6 IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1 beta is known to promote IL-6 secretion from the stimulated membrane apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1 beta stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF. Results: When Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1 beta increased LIF production. After treating the apical surface with IL-1 beta, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones. Conclusion: The site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.

Decreased NADPH oxidase expression and antioxidant activity in cachectic skeletal muscle

Background - Cancer cachexia is the progressive loss of skeletal muscle protein that contributes significantly to cancer morbidity and mortality. Evidence of antioxidant attenuation and the presence of oxidised proteins in patients with cancer cachexia indicate a role for oxidative stress. The level of oxidative stress in tissues is determined by an imbalance between reactive oxygen species production and antioxidant activity. This study aimed to investigate the superoxide generating NADPH oxidase (NOX) enzyme and antioxidant enzyme systems in murine adenocarcinoma tumour-bearing cachectic mice. Methods - Superoxide levels, mRNA levels of NOX enzyme subunits and the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidise (GPx) and catalase was measured in the skeletal muscle of mice with cancer and cancer cachexia. Protein expression levels of NOX enzyme subunits and antioxidant enzyme activity was also measured in the same muscle samples. Results - Superoxide levels increased 1.4-fold in the muscle of mice with cancer cachexia, and this was associated with a decrease in mRNA of NOX enzyme subunits, NOX2, p40phox and p67phox along with the antioxidant enzymes SOD1, SOD2 and GPx. Cancer cachexia was also associated with a 1.3-fold decrease in SOD1 and 2.0-fold decrease in GPx enzyme activity. Conclusion - Despite increased superoxide levels in cachectic skeletal muscle, NOX enzyme subunits, NOX2, p40phox and p67phox, were downregulated along with the expression and activity of the antioxidant enzymes. Therefore, the increased superoxide levels in cachectic skeletal muscle may be attributed to the reduction in the activity of endogenous antioxidant enzymes.

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension. © 2011 The Author(s).

NADPH oxidase signaling and cardiac myocyte function

The NADPH oxidase family of enzymes has emerged as a major source of reactive oxygen species (ROS) that is important in diverse cellular functions including anti-microbial defence, inflammation and redox signaling. Of the five known NADPH oxidase isoforms, several are expressed in cardiovascular cells where they are involved in physiological and pathological processes such as the regulation of vascular tone, cell growth, migration, proliferation, hypertrophy, apoptosis and matrix deposition. This article reviews current knowledge regarding the role of NADPH oxidases in cardiomyocyte function in health and disease. © 2009 Elsevier Inc. All rights reserved.

What checkers actually check:an eye tracking study of inhibitory control and working memory

Background - Not only is compulsive checking the most common symptom in Obsessive Compulsive Disorder (OCD) with an estimated prevalence of 50–80% in patients, but approximately ~15% of the general population reveal subclinical checking tendencies that impact negatively on their performance in daily activities. Therefore, it is critical to understand how checking affects attention and memory in clinical as well as subclinical checkers. Eye fixations are commonly used as indicators for the distribution of attention but research in OCD has revealed mixed results at best. Methodology/Principal Finding - Here we report atypical eye movement patterns in subclinical checkers during an ecologically valid working memory (WM) manipulation. Our key manipulation was to present an intermediate probe during the delay period of the memory task, explicitly asking for the location of a letter, which, however, had not been part of the encoding set (i.e., misleading participants). Using eye movement measures we now provide evidence that high checkers’ inhibitory impairments for misleading information results in them checking the contents of WM in an atypical manner. Checkers fixate more often and for longer when misleading information is presented than non-checkers. Specifically, checkers spend more time checking stimulus locations as well as locations that had actually been empty during encoding. Conclusions/Significance - We conclude that these atypical eye movement patterns directly reflect internal checking of memory contents and we discuss the implications of our findings for the interpretation of behavioural and neuropsychological data. In addition our results highlight the importance of ecologically valid methodology for revealing the impact of detrimental attention and memory checking on eye movement patterns.

The effect of growth conditions on the synthesis of Pseudomonas aeruginosa terminal oxidase.

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